Platelet-derived growth factor BB (PDGF) stimulates DNA synthesisthrough a mechanism that is at least partially dependent uponSrc family tyrosine kinases, although the signal transductionpathway downstream of Src is poorly understood. We have studiedthe signaling between Src and different protein kinase C (PKC)isoforms and its possible role in the regulation of PDGF-stimulatedDNA synthesis. We found that Src promoted the tyrosine phosphorylationof PKC, and its subsequent degradation. Enforced expressionof PKC inhibited PDGF-stimulated DNA synthesis, whereas expressionof PKC and PKC did not, a finding consistent with a model inwhich PKC negatively regulates G1-to-S-phase progression. Weused mutagenesis to map a critical Src phosphorylation siteon PKC to tyrosine 311. A mutant form of PKC in which tyrosine311 was replaced with phenylalanine (Y311F) was more stablein the presence of Src, suggesting that Src-induced degradationwas a direct result of PKC tyrosine phosphorylation. We concludethat PKC is downstream of Src but is unlikely to play a positiverole in the signaling pathway by which Src promotes DNA synthesis.

PDGF2
stimulates a mitogenic response in mesenchymally derivedcells such as fibroblasts as well as other cell types. The Srcfamily kinases Src, Fyn, and Yes have been implicated in PDGFsignaling by their association with the phosphorylated PDGFRand their subsequent activation (1, 2)
. Although there issome redundancy of function between these kinases, as a groupthey appear to be essential for PDGF-stimulated mitogenesisin fibroblasts. Inactivation of endogenous Src family kinaseswith either a neutralizing antibody or by expression of kinase-inactiveor SH3 domain-deleted forms of Src or Fyn inhibit PDGF-stimulatedDNA synthesis (3, 4, 5)
.

It is unlikely that Src kinases have only a single substrateinvolved in the regulation of mitogenesis. Src substrates possiblyinvolved in PDGF signaling include PKC(6)
, the PDGFR itself(7)
, Shc (8)
, Grb-2 (9)
, the inositol 1,4,5-triphosphate(IP3) receptor (10)
, the phosphatidylinositol 3'-kinase p85subunit (11, 12)
, and Eps8 (13)
. Here we focus on the roleof Src in PKC regulation and the possible role that PKC playsin the regulation of mitogenesis.

It has previously been reported that PKC serves as a substratefor Src (14)
. However, there is some disagreement about theeffect of tyrosine phosphorylation on PKC activity. Some reportssuggest that tyrosine phosphorylation activates PKC(14, 15)or modifies its substrate preference (16)
. Others report thattyrosine phosphorylation decreases PKC activity (17, 18)
.It should be noted that these apparently conflicting studiesdiffer in several key aspects, including whether they used purifiedPKC tyrosine phosphorylated in vitro or PKC extracted from stimulatedcells, the cell type, the nature of the stimulus, and whetherthey measured PKC activity directly or addressed changes inPKC activity in different cellular fractions.

Several reports have also suggested that PKC is involved inPDGF signaling. Li et al.(6)
note that in cells overexpressingboth PDGFR and PKC, PDGF causes an increase in PKC activityassociated with the membrane fraction, the exclusive locationof tyrosine-phosphorylated PKC. Translocation of PKC to themembrane fraction is also observed in PDGF-stimulated vascularsmooth muscle cells (19)
and fibroblasts (20)
. In addition,a kinase-inactive mutant of PKC inhibits cellular transformationby the sis proto-oncogene, which is closely related to PDGF(21)
. The results of Li et al.(21)
imply that PKC promotesPDGFR-stimulated anchorage-independent growth. This is somewhatat odds with other data concerning the effect of PKC. For example,kinase-active PKC has been shown to inhibit cell cycle progression.Watanabe et al.(22)
show that phorbol ester treatment of Chinesehamster ovary cells overexpressing PKC causes them to arrestin G2/M phase. Also, overexpression of PKC in either NIH3T3cells or human glioma cells reduces their rate of growth andthe density to which they grow (23, 24)
.

In the experiments described here, we have examined in moredetail the role of Src phosphorylation of PKC and its involvementin PDGF-mediated signal transduction.

Tyrosine Phosphorylation and Protein Instability of PKC in Src-transformed Cells.
We observed that PKC was highly tyrosine-phosphorylated in NIH3T3cells stably transfected with an activated mutant of Src (527cells), consistent with previous reports that PKC is a Src substrate.However, we also noted that the level of PKC was significantlylower in 527 cells compared with the parental NIH3T3 cells (Fig.1A)
. Treatment of NIH3T3 cells with PDGF, but not FCS, stimulateda modest increase in the tyrosine phosphorylation of endogenousPKC. This elevation of PKC tyrosine phosphorylation also correspondedwith a modest decrease in the level of PKC (Fig. 1A)
.

Fig. 1. Cells stably transfected with activated Src have diminished levels of PKC. A, NIH3T3 cells or NIH3T3 cells stably transfected with activated Src (527 cells) were quiesced in insulin/transferrin (24 h) and then stimulated with either 10% v/v FCS, 25 ng/ml PDGF, or 100 nM PMA for 10 min. The top panel shows an antiphosphotyrosine immunoblot of immunoprecipitated PKC. The bottom panel shows the same blot stripped and reprobed for the level of PKC. B, NIH3T3 cells and 527 cells were labeled with [35S]methione and cysteine, PKC immunoprecipitated, and resolved by 10% SDS-PAGE and autoradiography. C, PKC was immunoprecipitated from TGR-1 cells and TGR-1 cells stably transfected with the activated human Src SrcY530F, resolved on 10% SDS-PAGE, and visualized by anti-PKC immunoblotting.

The treatment of cells with phorbol esters, including PMA, ultimatelyleads to the degradation of phorbol ester- and diacylglycerol-sensitiveisoforms of PKC (25, 26)
. We observed a reduction in the levelof PKC after treatment with PMA, but unlike PDGF stimulation,PMA did not stimulate detectable tyrosine phosphorylation ofPKC in NIH3T3 cells. However, both PDGF and PMA increased thetyrosine phosphorylation of PKC in 527 cells. This is consistentwith previous studies in which activation of PKC renders ita better substrate for Src (14)
. It is also likely that phorbolester stimulates translocation of PKC to the membrane (27, 28), making it more accessible to Src.

The apparent reduction of the steady-state level of PKC in 527cells was not due to impaired detection of the tyrosine-phosphorylatedform because the anti-COOH-terminal antibody used to immunoprecipitatePKC was clearly able to recognize tyrosine-phosphorylated PKC(Fig. 1A)
, and similar experiments using an antibody raisedto a fusion protein of the NH2-terminal half of PKC also showeda lower level of PKC in 527 cells (data not shown). Furthermore,immunoprecipitation of 35S-labeled PKC, followed by detectionby autoradiography, also showed a decreased level of PKC in527 cells (Fig. 1B)
. In addition, treatment of tyrosine-phosphorylatedPKC with alkaline phosphatase did not increase the apparentlevel of PKC detected, despite reducing the phosphorylation(data not shown). Nor was the decreased level of PKC in 527cells a clonal artifact or NIH3T3 cell specific, because TGR-1cells [a fibroblastic cell line derived from Rat-1 (29)
] alsohad a lower level of endogenous PKC when stably transfectedwith an activated mutant of human Src (tyrosine 530 mutatedto phenylalanine; human SrcY530F; Fig. 1C
).

We considered the possibility that the decreased level of PKCdetected in Src-transformed cells might be due to its translocationto a detergent-insoluble fraction, although this seemed unlikelybecause even protein-denaturing extraction conditions (see "Materialsand Methods") failed to increase the level of PKC detected.If insolubility were the cause of the decreased level of PKC,its level would be diminished according to its distributionbetween the cell fractions, but the rate of protein turnoverwould be unaltered. We therefore compared the stability of PKCin NIH3T3 cells and 527 cells. PKC showed little sign of degradationover a 6-h period in NIH3T3 cells as determined by 35S pulsechase (Fig. 2A)
or using cycloheximide to inhibit protein synthesis(Fig. 2B)
. However, PKC was much less stable in 527 cells.Both 35S pulse-chase and cycloheximide treatment revealed anincreased rate of PKC turnover relative to that of the parentalNIH3T3 cells (Fig. 2, A and B)
. The half-life of PKC was estimatedas 3.5 h in 527 cells by quantitation of 35S autoradiography.We also asked whether Src affected the level of PKC mRNA. Fig.2C
shows a Northern blot comparing the level of PKC mRNA in527 cells and the parental NIH3T3 cells. We found no evidencethat expression of activated Src affects the PKC transcriptlevel. We therefore conclude that the level of PKC is reducedthrough increased degradation.

Fig. 2. Src promotes PKC degradation. A, NIH3T3 cells and 527 cells were labeled 16 h with [35S]methionine/cysteine and washed, and Cys/Met chase medium was added. At the times indicated, cells were lysed. PKC was immunoprecipitated and resolved on 10% SDS-PAGE and visualized by autoradiography. The PKC band was excised, and the level of 35S incorporation was quantitated by liquid scintillation counting. The results corrected for background counts are shown in the two graphs. The half-life of PKC in 527 cells is indicated on the graph to the right. B, NIH3T3 cells and 527 cells were treated with 20 µg/ml cycloheximide and then lysed at the times indicated. PKC was immunoprecipitated and resolved by SDS-PAGE and immunoblotted for the level of PKC. C, RNA from NIH3T3 cells and 527 cells was probed for the level of PKC mRNA. The bottom panel shows the ß-actin control. D, 527 cells were treated with the Src kinase inhibitor PP2 (5 µM) or the proteasome inhibitors Clasto-lactacystin-ß-lactone (c-lc; 1 µM) or MG132 (132; 10 µM) for 16 h, and then PKC was immunoprecipitated and resolved by SDS-PAGE, and its level was determined by immunoblotting.

A major route of protein degradation involves proteasomes, largeprotease complexes located in both the cytoplasm and the nucleus.Proteasomes are implicated primarily in the degradation of ubiquitinatedprotein but also in nonubiquitinated protein (30, 31, 32)
.To test the hypothesis that tyrosine phosphorylation promotesPKC degradation and to determine whether proteasomes were involved,we measured the effect of proteasome inhibitors on the levelof PKC in 527 cells. Clasto-lactacystin-ß-lactone, whichinhibits several different peptide hydrolytic activities ofthe proteasome (33)
, and MG132, which inhibits protein degradationby the proteasome without affecting its ATPase or isopeptidaseactivity (34, 35)
, both elevated the level of PKC in 527 cells(Fig. 2D)
. Similar results were seen for proteasome inhibitor1, which inhibits the chymotrypsin-like activity of the proteasome(Ref. 36
; data not shown). Together, these data suggest thatproteasomes participate in the degradation of tyrosine-phosphorylatedPKC.

Recently, Hanke et al.(37)
described the tyrosine kinase inhibitorsPP1 and PP2 and demonstrated that they were potent Src familykinase inhibitors, but only weakly inhibited ZAP-70 and JAK2.In further analysis of the selectivity of these compounds, wefound that they inhibited both PDGFR kinase activity and Srckinase activity with similar potency (PP1-Src IC50 = 0.4 µM,PP1-PDGFR IC50 = 1.6 µM, PP2-Src IC50 = 1.4 µM,and PP2-PDGFR IC50 = 1.5 µM), precluding us from usingthem to study the role of Src in PDGF signal transduction. However,we could use them to study Src-transformed cells. For example,we have observed that PP1 and PP2 reverse the transformed phenotypeof 527 cells, causing them to flatten and lose their characteristicactin rings.3
We examined the effect of PP2 on the tyrosinephosphorylation of PKC and on its protein level in 527 cells.PP2 both decreased the tyrosine phosphorylation of PKC and increasedits protein level, effectively reversing the effects of Srcon PKC (Fig. 3A)
. This supports the conclusion that tyrosinephosphorylation of PKC results in its instability.

Fig. 3. PP2 modulates the tyrosine phosphorylation, protein level, and activity of PKC. A, 527 cells were incubated for 16 h in the presence of 5 µM PP2 or DMSO alone as a control and then lysed. The level of PKC protein and the level of its tyrosine phosphorylation were determined by immunoprecipitation and immunoblotting. The left panel shows an antiphosphotyrosine blot. The right panel shows an anti-PKC blot. B, 527 cells overexpressing PKC (527 cells) were incubated for 16 h with PP2 (5 µM) or with DMSO alone and then lysed according to the PKC kinase protocol. PKC was immunoprecipitated from a serial dilution of each lysate (1.0, 0.5, 0.25, 0.125, and 0.0 mg/ml, indicated by the narrowing bar. The presence or absence of anti-PKC antibody in the immunoprecipitation is indicated by + or -, respectively. Each immunoprecipitate was subjected to PKC kinase assay using MBP as a substrate (top panel), and the level of PKC in each immunoprecipitate was determined by immunoblotting (bottom panel).

We next asked what effect tyrosine phosphorylation of PKC hadon its intrinsic kinase activity. We measured the PKC-associatedkinase activity in 527 cells overexpressing PKC (527 cells)that had been treated with PP2 or mock-treated with solventalone. Fig. 3B
shows the PKC kinase activity detected in decreasingconcentrations of cell lysate. Also shown are the levels ofPKC protein in each immunoprecipitate. Consistent with Fig.3A
, PP2 treatment increased the level of PKC (Fig. 3B
, bottompanel). Despite this, however, PKC kinase activity was lowerthan in the control cells in which PKC remains tyrosine-phosphorylated.The lower level of PKC activity was not caused by PP2 copurifyingwith and inhibiting PKC because it had no effect on PKC activitywhen added directly to the cell lysate as opposed to the preincubationwith intact cells (data not shown). We conclude that tyrosinephosphorylation of PKC by Src has two effects: (a) it increasesthe specific activity of PKC; and (b) it also causes the proteinto become unstable.

PKC and PDGF Stimulation.
We next measured the time course of PKC tyrosine phosphorylationand activity during stimulation of cells by PDGF. PKC underwentonly a brief period of tyrosine phosphorylation during the initial2030 min of PDGF stimulation of TGR-1 cells (Fig. 4A)or NIH3T3 cells (Fig. 1A)
. Tyrosine phosphorylation was followedby a modest decrease in the level of PKC in both cell types.PDGF also stimulated a brief period of activation (this is moreconvincing in TGR-1 cells, which express higher levels of PKCthan NIH3T3 cells; Fig. 4A
and data not shown) followed bya sustained decrease in PKC activity in both TGR-1 cells andNIH3T3 cells (Fig. 4, B and C)
. This pattern of tyrosine phosphorylation,activation, and decrease in protein levels is consistent withthe effects of PKC tyrosine phosphorylation in Src-transformedcells.

Fig. 4. PDGF stimulates a pulse of PKC tyrosine phosphorylation, activation, and a subsequent decrease in the PKC level. TGR-1 or NIH3T3 cells were quiesced for 24 h in 0.5% FCS and then stimulated with 25 ng/ml PDGF for the times indicated. A, TGR-1 cells were lysed, and PKC tyrosine phosphorylation (top panel) and protein level (bottom panel) were determined by immunoprecipitation and immunoblotting. B, TGR-1 cells were lysed, and the activity of PKC was determined according to the PKC kinase assay protocol using MBP as a substrate. C, quantitation of the activity of PKC in TGR-1 cells (n = 3) or NIH3T3 cells (n = 4) stimulated for various times with 25 ng/ml PDGF. The data are presented as the fold activation of PKC. The error bars represent the SE. Significant difference from the unstimulated kinase activity (P 0.05) is indicated by *. P 0.005 is indicated by **.

The degradation of PKC is probably part of the mechanism thatterminates the brief pulse of PKC activity, but what is itssignificance with regard to the mitogenic signal of PDGF? Oneway to address this is to maintain artificially high PKC levelsby ectopic expression from plasmids microinjected into cells.We used bromodeoxyuridine incorporation into newly synthesizedDNA to compare the effect of PKC, PKC, and PKC expression onPDGF-stimulated DNA synthesis. PKC expression caused a significantreduction in the number of cells entering S phase, whereas incells overexpressing PKC and PKC, the percentage of cells enteringS phase was unaltered (Fig. 5A)
. It appears that prolongedexpression and therefore prolonged elevation of PKC activityprevents or delays cells from proceeding from G1 to S phase.Although we do not know the function of the brief activationof PKC, if any, we suggest that its rapid termination is importantfor PDGF-stimulated mitogenesis.

Fig. 5. Enforced expression of PKC inhibits PDGF-stimulated DNA synthesis. A, NIH3T3 cells were quiesced for 24 h in 0.5% FCS and then microinjected with plasmids (100 µg/ml) expressing either PKC, PKC, or PKC. After 16 h, cells were stimulated with 25 ng/ml PDGF in the presence of BrdUrd for 24 h and then fixed and stained for the expression of the PKC isoform and the incorporation of BrdUrd into the nuclear DNA. The data are presented as the percentage of cells that stained positively for BrdUrd incorporation (% BrdUrd; n 3). B, NIH3T3 cells were quiesced and then microinjected with pBP PKC (200 µg/ml) either alone or in combination with pSGT SrcY527F (25 µg/ml), stimulated with PDGF, and stained for PKC as described above. C, NIH3T3 cells were quiesced and then microinjected with pBP PKCY311F (200 µg/ml) either alone or in combination with pSGT SrcY527F (25 µg/ml), stimulated with PDGF, and stained for PKC as described above or for Src using monoclonal antibody EC10 when pSGTSrcY527F was the sole injected plasmid. Significant differences between uninjected and injected PDGF-stimulated cells are indicated by * for P 0.05 and ** for P 0.005 (n = 3). A significant difference between PDGF cells injected with PKC alone and PDGF-stimulated cells injected with both PKC and SrcY527F is indicated by , indicating P 0.05.

If Src mediates the down-regulation of PKC, overexpression ofactivated Src might be expected to rescue the inhibition ofDNA synthesis by PKC. As before, PKC inhibited PDGF-stimulatedDNA synthesis. Coexpression of activated Src with PKC causeda significant elevation in the number of PDGF-stimulated cellsentering S phase, consistent with a rescue of the PKC blockby Src (Fig. 5B)
. It should also be noted that coexpressionof this amount of activated Src with PKC caused a smaller butsignificant elevation of the number of unstimulated cells enteringS phase.

Analysis of PKC Mutants for Tyrosine Phosphorylation, Stability, and Function.
Several studies have sought to identify sites of tyrosine phosphorylationon PKC. Szallasi et al.(38)
demonstrated that mutation oftyrosine 52 reduced IgE antigen-induced tyrosine phosphorylation.Li et al.(39)
identified tyrosine 187 as a phosphorylationsite. Konishi et al.(40)
demonstrated that mutation of tyrosines512 and 523 within the kinase domain abolished enzyme activation,but they noted that the mutated enzyme was still phosphorylatedto some extent in response to H2O2. To identify other sitesphosphorylated by Src, we generated a series of PKC tyrosineto phenylalanine (YF) point mutants targeting conserved tyrosineswith high surface probability and NH2-terminal acidic groups,because these features are often seen in peptide substratesof tyrosine kinases (41)
. The YF point mutations included:(a) Y311F, which lies in the extended region between the cysteine-richdomains and the kinase domain; (b) Y372F, which lies in thesmall lobe of the kinase domain; (c) Y565F, which lies in thelarge lobe of the kinase domain; and (d) Y628F, which lies inthe COOH-terminal tail. We tested the ability of Src to phosphorylatethe PKC YF mutants by coexpressing them with activated Src inHEK293 cells (Fig. 6A)
and assaying the level of PKC tyrosinephosphorylation. Although we often saw a slight reduction inthe level of tyrosine phosphorylation of several mutants relativeto the wild type, PKCY311F was unique in that it was not a substratefor Src, with no phosphorylation over the background being detected.It is interesting to note that the sequence surrounding tyrosine311 shows the highest similarity to the optimal Src substratesequence predicted by Songyang et al.(42)
.

Fig. 6. Mutation of PKC tyrosine 311 prevents its phosphorylation by Src and renders it resistant to Src-induced degradation. A, tyrosine to phenylalanine point mutations of PKC were generated and tested for their ability to act as Src substrates. HEK293 cells were transfected with 4 µg of each PKC mutant (identified by the residue number mutated, e.g., 311 indicates PKC Y311F) or wild type (WT) in pBP, in combination with 4 µg of pBP SrcY530F or 4 µg of pBP as a control. After 16 h, cells were lysed, and the level of PKC tyrosine phosphorylation was determined by immunoprecipitation and antiphosphotyrosine immunoblotting (top panel). The bottom panel shows the immunoblot for the level of PKC. B, 527 cells overexpressing PKC (527 cells) were lysed in 1 ml of RIPA containing no protease inhibitors. The lysate was incubated at 37°C with 100 µl of agarose immobilized trypsin (Sigma). At the times indicated, the trypsin-agarose was pelleted, and a 250-µl aliquot of lysate was removed, and 1 mM PMSF was added. An antibody raised to the COOH-terminal sequence of PKC was used to immunoprecipitate protein from each aliquot, and phosphotyrosine-containing protein was detected by SDS-PAGE and immunoblotting (left panel). The same experiment was performed comparing 527 (5) and 527 () cell lysates that were either digested or not digested with trypsin-agarose for 60 min. C, HEK293 cells were transfected with i) 8 µg of pBP PKC WT + 0.4 µg of pBP, ii) 8 µg of pBP PKC Y311F + 0.4 µg of pBP, iii) 8 µg of pBP PKC WT + 0.4 µg of pBP SrcY530F, or iv) 8 µg of pBP PKC Y311F + 0.4 µg of pBP SrcY530F. After 16 h, each set of transfected cells was trypsinized and divided equally into a 6-well plate. After an additional 12 h, 20 µg/ml cycloheximide was added. Cells were scraped, washed, and lysed at the times indicated after the addition of cycloheximide. The level of PKC was determined by immunoprecipitation and immunoblotting. D, HEK293 cells were transfected with either 8 µg of pBP PKC WT or pBP PKC Y311F for 16 h and trypsinized, and each was divided equally into a 6-well dish. After 12 h, cells were treated with either 100 nM PMA or DMSO alone. Cells were scraped, washed, and lysed at the times indicated after the addition of PMA. The level of PKC was determined by immunoprecipitation, SDS-PAGE, and immunoblotting.

Fig. 6A
also shows that, just as in NIH3T3 cells, the levelof endogenous PKC in HEK293 (human embryo renal cortical) cellsis significantly reduced when activated Src is expressed [comparethe level of endogenous PKC in the control mock-transfectedHEK293 cells in the far left lane of the bottom panel of Fig.6A
with the level of endogenous PKC in cells transfected withactivated Src in Lane Src - (seventh from the left)]. Even whenhighly expressed by transient transfection in HEK293 cells,PKC shows a Src-dependent decrease in its protein level in thepresence of cycloheximide (Fig. 6C)
, suggesting that Src-inducedPKC degradation is not specific to fibroblastic cell lines.

As part of our study of the tyrosine phosphorylation sites ofPKC, we also performed a series of experiments designed to detecttyrosine phosphorylation of COOH-terminal fragments of PKC.Lysates of 527 cells were digested with immobilized trypsin,and then fragments containing the COOH terminus were isolatedby immunoprecipitation using an antibody raised to the extremeCOOH-terminal sequence of PKC. The fragments were analyzed byantiphosphotyrosine immunoblot (Fig. 6B)
. Digestion with trypsinover a period of 60 min reduced the 7883-kDa PKC bandto a trypsin-resistant fragment of 20 kDa. We were concernedabout whether this fragment was really derived from PKC, andnot from another protein with an epitope recognized by the COOH-terminalantibody that was exposed when digested with trypsin. We addressedthis by comparing the level of the 20-kDa fragment in 527 cellswith that of 527 cells overexpressing PKC (Fig. 6B)
. Comparisonof the level of the 20-kDa protein in the two right lanes ofFig. 6B
shows that trypsinization of the lysate from cellsoverexpressing PKC produced an elevated level of the 20-kDaprotein relative to the parental cells. This strongly suggeststhat the 20-kDa fragment is indeed derived from PKC. This 20-kDafragment contains both the COOH terminus and at least one siteof tyrosine phosphorylation. A COOH-terminal fragment containingtyrosine 311 would be 42 kDa in size. Even allowing for aberrantSDS-PAGE mobility, it is unlikely that the COOH-terminal trypsin-resistant20-kDa fragment contains tyrosine 311, suggesting that additionaltyrosine phosphorylation sites exist. The fact that a singlemutation was capable of blocking all detectable phosphorylationof PKC by Src and the experiments described above are consistentwith a mechanism of processive phosphorylation beginning withtyrosine 311.

We have presented evidence that Src promotes the degradationof PKC. Although PKC is a Src substrate, it is not certain thatits tyrosine phosphorylation is directly responsible for itsdegradation. The initiation of degradation may occur via anindirect mechanism involving either diacylglycerol, the originalsecond messenger shown to activate PKC isoforms, or phosphatidylinositol-3,4-P2or phosphatidylinositol-3,4,5-P3, which were identified morerecently as second messengers that activate the PKC isoforms, , and . We do not believe that phosphatidylinositol-3,4-P2or phosphatidylinositol-3,4,5-P3 is involved in the Src-induceddegradation of PKC because the phosphatidylinositol 3'-kinaseinhibitor LY294002 (25 µM) had no effect on PKC levelsin Src-transformed cells (data not shown), but this does notdisprove the involvement of a second messenger. The fact thatSrc was unable to phosphorylate PKCY311F enabled us to testwhether Src is required to phosphorylate PKC to promote itsdegradation. The stability of PKC WT or PKCY311F was comparedin the presence or absence of activated Src, using cycloheximideto inhibit protein synthesis in transfected HEK293 cells. Toavoid fluctuations in PKC levels due to differences in transfectionefficiency, each time course of cycloheximide treatment wasperformed on a single transfected sample divided equally betweentime points. Transiently expressed PKC WT and PKCY311F werestable over the 4-h experiment in the presence of cycloheximide.When cotransfected with activated Src, PKC WT was rapidly degraded(Fig. 6C)
in a manner similar to that seen for endogenous PKCin cells stably transfected with activated Src (Fig. 2B)
. Incontrast, PKCY311F showed no sign of degradation when cotransfectedwith activated Src. We conclude that Src-induced degradationof PKC is a direct result of its tyrosine phosphorylation. Wewere curious to see whether PKCY311F was also resistant to degradationinduced by phorbol esters, so we tested the effect of PMA onHEK293 cells transiently transfected with PKC WT or PKCY311F.Both PKC and PKCY311F were stable over the 8-h period of theexperiment when treated with DMSO alone as a control. However,PMA (100 nM) reduced the level of both PKC WT and PKCY311F within4 h of treatment (Fig. 6D)
. Therefore, mutation of tyrosine311, although able to protect PKC against Src-induced degradation,does not protect it against the degradation elicited by PMAtreatment.

Having shown that coexpression of an activated mutant of Srcis able to rescue the inhibition of PDGF-stimulated DNA synthesisby PKC (Fig. 5B)
, we went on to test whether this was alsotrue for PKCY311F. Fig. 5C
shows the proportion of NIH3T3 cellsgoing through PDGF-stimulated S-phase entry after the expressionof SrcY527F and PKCY311F. As in Fig. 5B
, expression of SrcY527Fcaused a slight elevation of DNA synthesis in unstimulated cells.However, SrcY527F did not cause an additional increase in DNAsynthesis in PDGF-stimulated cells. PKCY311F was similar toPKC WT in that its expression inhibited PDGF-stimulated DNAsynthesis. However, coexpression of SrcY527F did not show anysignificant rescue of the inhibition of DNA synthesis by PKCY311F,suggesting that the mechanism by which Src rescues the PKC blockrequires the tyrosine phosphorylation of PKC at tyrosine 311.

Despite the abundance of information already obtained on Srcand many of its substrates, the mechanism by which Src mediatesits mitogenic effect is not yet clear. We have focused on oneof the Src substrates, PKC, to determine whether it is partof the PDGF signal transduction pathway downstream of Src thatis required for DNA synthesis. Our data implicate PKC in thispathway but do not support a simple positive kinase cascademodel. Src activates PKC but promotes its degradation, and sustainedexpression of PKC inhibits DNA synthesis.

One model that fits these data predicts that Src negativelyregulates the level of PKC, a negative regulator of DNA synthesis,thereby promoting a mitogenic signal. However, it is probablynot that simple because Src also transiently activates PKC.It is also clear that PKC activation and the fall in its proteinlevel follow a tightly regulated temporal pattern during thevery early stages of PDGF stimulation. As an aside, other stimuliunrelated to mitogenesis have also been shown to activate apulse of PKC activity. The calcium-mobilizing agents carbacholand substance P stimulate a pulse of PKC activity in salivarygland parotid acinar cells (43)
. It is not certain whetherit is the initial activation of PKC or the cessation of itssignal that is important for mitogenesis. The fact that sustainedexpression of PKC inhibits cell growth suggests the latter hypothesis.Whereas this may represent a real antimitogenic function, wemust also consider the possibility that it is due to inappropriateexpression of PKC. However, the fact that neither PKC nor PKCexpression was inhibitory suggests some specificity of the effect.Others have also described a negative effect of PKC on cellgrowth, but with some important differences. Watanabe et al.(22)
observed cell cycle arrest in G2-M phase, whereas ourdata demonstrate a block in G1-S phase. This difference couldbe due to the different stimuli being used. Watanabe et al.examined the effect of TPA treatment, whereas our data applyexclusively to PDGF stimulation. Another study based on thepharmacological effects of TPA and bryostatin-1 suggests thatPKC has an antimitogenic function. Lu et al.(44)
demonstratethat bryostatin-1, which has complex effects on PKC includingthe inhibition of TPA-induced down-regulation of PKC in cellsoverexpressing c-Src, blocks the tumor-promoting effects ofTPA. It is also likely that one of the other PKC isoforms mediatesa positive role in PDGF-stimulated mitogenesis. A series ofdominant inhibitory PKC mutants with broad specificity haverecently been described (45)
. Expression of these forms ofPKC, PKC, and PKC inhibit PDGF-stimulated DNA synthesis,4
suggestingthat broad inhibition of PKC activation results in a mitogenicblock.

The mechanism of PKC regulation has important implications forcell growth and might be expected to involve multiple levelsof control and be regulated by converging signaling pathways.Olivier et al.(46)
demonstrate that transforming growth factorß1 selectively blocks bombesin-induced down-regulationof PKC within S phase. Shih et al.(47)
reported that TPA regulatesPKC expression by down-regulating its mRNA both transcriptionallyand posttranscriptionally. However, we found no evidence thatSrc affects the level of PKC mRNA. Rather, our evidence implicatesSrc in the degradation of PKC. This adds to the growing bodyof evidence that tyrosine phosphorylation of PKC activates itbut renders it highly susceptible to degradation. Recently,Zang et al.(18)
demonstrated that the viral homologue of Src,v-Src, is able to form a complex with PKC that becomes phosphorylatedon tyrosine. Whereas Zang et al.(18)
noted a decrease in PKCactivity, they did not address its cause or whether PKC proteinlevels were altered. The effect of proteasome inhibitors onthe level of PKC in Src-transformed cells implicates a proteasome-dependentmechanism of degradation. Proteasomes were originally shownto degrade ubiquitinated proteins (30)
, although nonubiquitinatedproteins are now known to be degraded by this mechanism (31, 32). We do not believe that the effect of the proteasome inhibitorson PKC is due to nonspecific activity because structurally andmechanistically distinct compounds have the same effect, increasingthe level of PKC in Src-transformed cells. Although we wereunable to convince ourselves (using antiubiquitin immunoblotting)that PKC is ubiquitinated after its tyrosine phosphorylation,we do not exclude this possibility. Recently, Lu et al.(48)demonstrated that activation of PKC, PKC, or PKC by phorbolesters triggers their ubiquitination and degradation. We frequentlyobserved a tyrosine-phosphorylated 100110-kDa proteinin PKC immunoprecipitates from Src-transformed cells, but thisis unlikely to represent polyubiquitinated PKC because it doesnot have the characteristic ladder of bands normally associatedwith ubiquitination. However, it could represent monoubiquitination.PKC may also be degraded by a mechanism involving the interleukin1ß-converting enzyme-like proteases because it has twoDXXD consensus caspase-3 cleavage sites (49)
.

The fact that a single point mutation (Y311F) of PKC preventsits phosphorylation by Src suggests that Src uses a mechanismof processive phosphorylation. Such a mechanism has been implicatedin the sequential phosphorylation of the chain by Lck and thephosphorylation of p130cas by Abl. The effect of SH2 mutationssuggests that processive phosphorylation proceeds via an initialhigh-affinity phosphorylation site to which the kinase bindsusing its SH2 domain, enabling subsequent phosphorylation oflower affinity sites (50, 51)
. It is interesting to note thataccording to the prediction of the optimal substrate sequenceof Src (AEEEIYGEFEAKKKK) by Songyang et al.(42)
, the sequencesurrounding tyrosine 311 (ETVGIYQGFEKKTAV) suggests it is thehighest affinity Src phosphorylation site on PKC. However, thesequence bears little resemblance to the optimal Src SH2 bindingsequence PQYEEI (52)
, raising the question of whether a thirdadaptor protein is involved. This SH2 model of processive phosphorylationdoes not exclude the possibility that tyrosine phosphorylationof Y311 changes the conformation of PKC, exposing other phosphorylationsites.

We have not addressed the possibility that tyrosine phosphorylationof PKC modifies its substrate preference because the specificsubstrates involved in the antimitogenic effect need to be characterized.Also, we recognize that different stimuli, even though theyresult in PKC tyrosine phosphorylation, may ultimately affectits activity in different ways, depending on other modes ofregulation. PKC was recently shown to be regulated by PDK1 ora homologue (53)
and is likely to be controlled in a mannersimilar to PKC, by multisite phosphorylation (54, 55, 56)
.

Some PKC substrates are common substrates for several PKC isoformsand thus are unlikely to mediate a PKC-specific effect suchas inhibition of cell growth. These include the myristoylatedalanine-rich PKC-kinase substrate MARCKS, which is sequentiallyphosphorylated by conventional, novel, and atypical isoformsof PKC (57)
and the cytoskeletal protein vimentin (58)
. However,the elongation factor eEF-1 is a more exclusive PKC substrate(59)
. This protein has been found in complexes containing ribosomalRNA, aminoacyl-t-RNA, and ribosomal protein L8, which was identifiedas a PKC-associating protein by yeast 2-hybrid (60)
. PerhapsPKC affects cell cycle progression through the regulation ofprotein synthesis. Alternatively, eEF-1 has also been implicatedin the regulation of cytoskeletal structures (61, 62)
.

The dual effects of Src on PKC, its activation and its degradation,might go some way to explaining the conflicting reports of theeffect of tyrosine phosphorylation on PKC activity. Phosphorylationof PKCin vitro will not have the complications of its degradation.Cellular studies not only deal with degradation, but also withwhat appear to be quite rapid kinetics of activation and inactivation.

There is considerable evidence implicating PKC in the regulationof mitogenesis and as a downstream target of Src during PDGFstimulation of fibroblastic cell lines. However, it is not possibleto place PKC in a simple model that invokes a positive kinasecascade leading from Src to the regulation of DNA synthesis.We have demonstrated that the phosphorylation of PKC by Srcrequires the tyrosine at position 311. Mutation of this siteprevents PKC from acting as a Src substrate, protects it fromphosphorylation induced-degradation, and prevents Src from rescuingthe inhibition of DNA synthesis by PKC. The antimitogenic effectsof PKC lead us to speculate that its degradation may contributeto cell cycle progression.

Antibodies.
The principal anti-PKC used for immunoblotting was a mouse monoclonalIgG2b raised against amino acids 114289 of human PKC,spanning the cysteine-rich repeats (Transduction Laboratories).The principal antibody used to immunoprecipitate PKC and forimmunofluorescent staining was an affinity-purified rabbit polyclonalantibody raised against a peptide corresponding to amino acids657673, mapping within the extreme COOH terminus of ratPKC. Similar antibodies raised to the COOH-termini of PKC andPKC were also used (Santa Cruz Biotechnology). Another anti-PKCantibody also used for immunoprecipitation was a rabbit polyclonalantibody raised against a GST fusion of amino acids 1302of rat PKC (Zymed). Antiphosphotyrosine mouse monoclonal (IgG2bk)4G10 and avian Src-specific monoclonal antibody EC10 were fromUpstate Biotechnology, and anti-BrdUrd mouse monoclonal wasfrom Boehringer Mannheim.

Plasmids.
Rat PKC, PKC, and PKC were subcloned into pBabePuro3 for stabletransfection and microinjection. Site-directed mutagenesis ofPKC was performed using the Quickchange site-directed mutagenesiskit (Stratagene). The Y311F mutation was made using oligonucleotideCGAATCCCTGGAATATTCCGACAGTC and its reverse complement. The Y372Fmutation was made using oligonucleotide CAAGGAAAGGTTCTTTGCAATCAAGTACCand its reverse complement. The Y565F mutation was made usingoligonucleotide GGACACACCACACTTCCCGCGCTGG and its reverse complement.The Y628F mutation was made using the oligonucleotide GAAATCCCCTTCAGACTTCAGCAACTTTGACCCAGAGand its reverse complement. Mutagenesis was verified by sequencing.An Eco47III/PinA1 fragment encompassing the Y311F mutation andPinA1/NdeI fragments encompassing the Y372F, Y565F, and Y628Fmutations were each subcloned back into wild-type PKC, and theinserts were sequenced to check for PCR errors.

Compounds.
Human recombinant PDGF was from Upstate Biotechnology. Proteasomeinhibitor, MG132 (carbobenzoxy-L-leucyl-L-leucyl-L-leucinal),clasto-lactacystin ß-lactone, and LY294002 were from Calbiochem).PP2 was provided by Sugen Inc. Cycloheximide and PMA were fromSigma. All other reagents were of analytical grade.

Microinjection and Immunofluorescent Staining.
Microinjection was performed with an Eppendorf Micromanipulator5171 and Transjector 5246 using borosilicate thin wall capillarieswith filament pulled on a Sutter Instrument Co. model P-97 Flaming/Brownmicropipette puller. Cells were seeded at 30% confluence oncoverslips, grown for 24 h, and then quiesced in 0.5% FCS andDMEM for 24 h before nuclear microinjection of the expressionplasmids of interest (plasmid concentrations ranged between40 and 200 µg/ml). After a period of 16 h to enable proteinexpression, cells were stimulated with 25 ng/ml PDGF in thepresence of 20 µM BrdUrd for 24 h, fixed using methanol/acetone(1:1; room temperature), treated with 2 M HCl at 37°C for15 min, and then washed twice with PBS. Cells were stained witha 1:40 dilution [in PBS (pH 7.6)] of affinity-purified rabbitanti-PKC isoform-specific antibodies (either anti-PKC, anti-PKC,or anti-PKC, depending on the isoform being expressed) or EC10anti-avian Src for 40 min, washed once in PBS, stained witha 1:40 dilution of Oregon Green-conjugated antirabbit IgG orTexas-Red-conjugated antimouse IgG (Molecular Probes), washedonce in PBS, stained with a 1:10 dilution of anti-BrdUrd monoclonalantibody, washed once with PBS, stained with a 1:25 dilutionof Texas Red-conjugated AffiniPure goat antimouse IgG (JacksonImmunoresearch Laboratories) in PBS containing 1 µg/mlbisbenzimide (Sigma), washed once with PBS and once with deionizedwater, and then mounted Biomeda Gel/Mount (MO1). In the caseof probing for Src using EC10, a directly conjugated fluorescein-anti-BrdUrdwas used to detect de novo DNA synthesis. A C-Imaging 640 imagingsystem was used to store and analyze images of cells expressingthe proteins of interest and to quantitate the proportion ofcells staining positive for BrdUrd incorporation into the DNA.

Transient Transfection.
HEK293 cells were transfected using 10 µl of LipofectAMINEand 0.510 µg of DNA/10-cm dish in Optimem over16 h and then either lysed in RIPA for immunoprecipitation ortrypsinized and replated at equal density in 6-well plates todetermine PKC stability. The quantity of DNA used in each transfectionis described in the figure legend.

35S Labeling of Cells and Pulse Chase.
Cells were labeled with 13 mCi of 35S Met/Cys (DuPontNew England Nuclear) for 16 h in Cys/Met-free MEM and 10% dialyzedFCS and then either prepared for immunoprecipitation or washedin PBS and chase medium containing 1.52 mg/ml methionine, 2.4mg/ml cysteine, and 10% FCS in DMEM. PKC was immunoprecipitatedfrom RIPA lysate, denatured in 2% SDS, diluted 20-fold in SDS-freeRIPA, reimmunoprecipitated, and then prepared for SDS-PAGE andautoradiography.

Northern Blotting.
RNA was prepared using the FastTrack 2.0 kit. Nothern blottingof PKC mRNA was performed using a 32P-labeled Eco47III/PinA1fragment from pBP PKC.

We thank Jianming Wu for help with the Northern blots, JohnSedivy for supplying us with the TGR-1 cells, Rich Cutler forreview of the manuscript, and all members of the Courtneidgelaboratory for helpful suggestions throughout the course ofthis work.

Footnotes

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